This application for a small grant (R03) requests support for research involving the development of a method to be used subsequently in research on the neuronal mechanisms mediating the development of age-related impairments in cognitive functions and the associated decline in the regulation and integrity of the basal forebrain (BF) cholinergic system. Disruption of nerve growth factor (NGF) signaling via tyrosine kinase receptor (TrkA) receptors has been hypothesized to result in the dysregulation of the BF cholinergic system and in impairments of the cognitive functions mediated via this major cortical input system. The first specific aim focuses on the construction and testing, in vitro and in vivo, of viral-mediated RNA interference (RNAi) to suppress the expression of TrkA receptors by BF cholinergic neurons. The initial experiments will involve the construction of plasmids designed to express TrkA microRNA (miRNA) under the control of a RNA polymerase II promoter, and screening of TrkA miRNA sequences for suppression of TrkA receptor expression in rat pheochromocytoma cells. Based on the screening of several plasmids in vitro, we will construct recombinant adeno-associated virus (AAV) vectors carrying plasmids expressing RNA polymerase II driven TrkA miRNA for in vivo gene delivery in rat BF neurons. Constructs will be infused into the BF of rats and viral transfection of BF cholinergic neurons and TrkA suppression will be evaluated. As non-cholinergic neurons do not express TrkA receptors, transfection of non-cholinergic neurons is expected to remain ineffective. Several control procedures will be employed to determine potential off-target/non-specific effects in cholinergic neurons. A second series of experiments will test the hypothesis that chronic silencing of BF TrkA receptors result in the attenuation of the capacity of cortical cholinergic inputs to release acetylcholine (ACh). Initial experiments will employ choline-sensitive microelectrodes to determine attenuated potassium-evoked ACh efflux in the cortex of rats exhibiting suppression of TrkA receptor expression. The final experiments will assess the cognitive consequences of persistent TrkA receptor expression. As attentional performance has been extensively demonstrated to indicate the status of cortical cholinergic neurotransmission, persistent suppression of TrkA receptor expression is hypothesized to result in enduring impairments in attentional performance, specifically a decrease in the animal's ability to detect attention-triggering cues. The expected results will form the basis for future research on the general hypothesis that developmental disruption of TrkA receptor signaling represents a major variable in the manifestation of age-related dysregulation of BF cholinergic neurons and associated impairments in cognitive functions. Moreover, the methods developed as part of this project will be of potential use for research aiming at silencing the expression of other neuronal gene products in vivo. PUBLIC HEALTH RELEVANCE The expected results of this research proposal will allow the development of a new method that can subsequently be used in exploring molecular mechanisms underlying age-related impairments in cognitive functions associated with mild cognitive impairment (MCI) and Alzheimer's disease. [unreadable] [unreadable] [unreadable]